Chronic pain causes debilitation and suffering in millions of Americans. Treatments to provide relief from this chronic pain are often ineffective. A critical and rate-limiting step is to prevent the transition from acute to chronic pain, but the molecular mechanisms underlying this process are poorly understood. Our application is targeted directly at filling this void. We recently discovered that low G protein-coupled receptor kinase 2 (GRK2) is a major factor that promotes transition to chronic hyperalgesia. Inflammation causes a reduction in GRK2 in pain transmitting neurons (nociceptors), an effect that can be mimicked in a mouse model using Cre-Lox technology. We recently demonstrated that low nociceptor GRK2 markedly prolongs hyperalgesia/allodynia induced by prostaglandin E2 (PGE2) and epinephrine without affecting baseline pain sensitivity. This project aims at understanding how inflammation causes a long-term reduction of GRK2 in nociceptors and at unraveling the molecular pathways that underlie how reduced GRK2 promotes the transition to chronic pain. Our hypothesis is that the inflammation-induced reduction in nociceptor GRK2 regulates signaling in response to cAMP-inducing mediators like PGE2. Low GRK2 causes this signaling to switch from protein kinase A towards the cAMP target known as "exchange protein directly activated by cAMP" (Epac) and its downstream targets. To test our hypothesis, we will answer three specific questions: 1. When and how does inflammation reduce GRK2 and increase Epac1 in nociceptors? 2. How do GRK2 and Epac1 interact to regulate Epac signaling to its downstream effectors? and 3. Can we generate proof of principle that targeting GRK2/Epac prevents transition to chronic pain? In aim 1, we will use immunofluorescence, stereology and qPCR analysis of nociceptor GRK2/Epac in vivo and in vitro to strengthen the emerging evidence for a critical role of the GRK2/Epac interface in the transition to chronic pain. In aim 2, we will use in vitro approaches with GRK2 deletion and kinase-dead mutants, kinase assays and co-immunoprecipitation to dissect underlying mechanisms. In aim 3, a viral overexpression system and classic genetic approaches will be used to generate novel mouse models with nociceptor-specific deletion of Epac1 and overexpression of GRK2. This aim will permit us to obtain the ultimate proof for the proposed role of GRK2 and EPAC in preventing the transition to chronic pain. This research application is innovative for three major reasons: (a) GRK2 is the first discovered endogenous inhibitor of Epac activation;(b) We will directly test our hypothesis that this newly discovered GRK2/Epac interface functions as an intracellular molecular switch that regulates cAMP-mediated hyperalgesic signaling and the transition to chronic pain;(c) Targeting the GRK2/Epac interface creates new possibilities for pharmacological interventions to prevent the transition from acute to chronic pain that avoids the disadvantages of targeting upstream receptors. This contribution is significant because it addresses the pathophysiology of a clinically relevant condition with the potential to identify new targets for drug discovery. PUBLIC HEALTH RELEVANCE: A novel molecular switch regulating transition from acute to chronic pain Project Narrative Chronic pain affects more than 50 million Americans every year, and pain relief for most patients is limited. The proposed research is relevant and significant to public health because it focuses on unraveling why pain does not resolve. We recently discovered that the interaction of two proteins, GRK2 and Epac, is very likely to regulate the transition from acute to chronic pain. Here we will characterize the molecular mechanisms by which these two proteins are involved in the transition from acute to chronic pain. Our results are expected to enable development of novel therapeutic strategies aimed at preventing the transition to chronic pain. The proposed research responds directly to PA-10-006, "Mechanisms, Models, Measurement and Management in Pain Research," that is sponsored by 12 of the 27 institutes and centers that compose the National Institutes of Health. More specifically, our results are likely to have a major impact on the mission of the National Institute of Neurological Disorders and Stroke that is to reduce the burden of neurological disorders, including chronic pain.